WO2021080400A1 - Polypeptide dérivé de l'oenanthe javanica et composition pharmaceutique le comprenant - Google Patents

Polypeptide dérivé de l'oenanthe javanica et composition pharmaceutique le comprenant Download PDF

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WO2021080400A1
WO2021080400A1 PCT/KR2020/095011 KR2020095011W WO2021080400A1 WO 2021080400 A1 WO2021080400 A1 WO 2021080400A1 KR 2020095011 W KR2020095011 W KR 2020095011W WO 2021080400 A1 WO2021080400 A1 WO 2021080400A1
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virus
polypeptide
seq
amino acid
parsley
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PCT/KR2020/095011
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English (en)
Korean (ko)
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주성수
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강릉원주대학교 산학협력단
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Publication of WO2021080400A1 publication Critical patent/WO2021080400A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a polypeptide consisting of a specific nucleotide sequence derived from parsley and a pharmaceutical composition comprising the polypeptide.
  • Immunity refers to a reaction that maintains the homeostasis of the body by discriminating between self and non-self in the human body and by recognizing and removing harmful substances that occur naturally in the human body or from outside.
  • the immune response is important in resisting harmful bacteria such as viruses, bacteria, and parasites that have invaded the human body from the outside, and in resisting or removing cancer cells generated inside.
  • Congenital immunodeficiency is a disease in which immune cells such as B cells and T cells do not exist from the original, and can be treated only with treatment methods such as gene therapy, antibody injection or bone marrow transplantation.
  • acquired immunodeficiency is that the immune component itself exists, but an abnormality occurs in the immune response process exhibited by them, and the state of the disease can be improved by enhancing the function of these components.
  • Korean Patent Laid-Open No. 10-2006-0047447 discloses monoacetyldiacylglycerol compounds represented by a specific formula for various immune systems. Disclosed is that it can be used for the treatment of diseases caused by deterioration of the function of, or various diseases such as cancer, arthritis, atopy, and dementia.
  • An object of the present invention is to provide a polypeptide composed of a specific amino acid sequence, an expression vector expressing the polypeptide, and a host cell transfected with the expression vector.
  • Another object of the present invention is to provide an immunomodulatory or antiviral use of the polypeptide of the present invention.
  • the present invention provides a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2.
  • the present invention provides an expression vector comprising a polynucleotide encoding the polypeptide.
  • the present invention provides a host cell transfected with the expression vector.
  • the present invention provides an immunomodulatory agent comprising a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 or a polynucleotide encoding the polypeptide as an active ingredient.
  • the present invention provides an immunomodulatory method comprising administering to an individual a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 or a polynucleotide encoding the polypeptide.
  • the present invention provides a use of a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 or a polynucleotide encoding the polypeptide for use in the manufacture of a medicament for immunomodulation.
  • the present invention provides an antiviral agent comprising a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 or a polynucleotide encoding the polypeptide as an active ingredient.
  • the present invention provides a method for preventing, improving, or treating a viral infection comprising administering to an individual a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 or a polynucleotide encoding the polypeptide.
  • the present invention provides a use of a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 or a polynucleotide encoding the polypeptide for use in the manufacture of a medicament for the prevention, amelioration or treatment of viral infection.
  • the polypeptide according to the present invention can be usefully used as an immunomodulatory agent by increasing the expression of various genes involved in the immune response in the body by a TLR signaling mechanism, and in particular, expression of genes that exhibit antiviral activity during the immune response. It can be usefully used as an antiviral agent by promoting it.
  • FIG. 1 is a diagram showing the results of confirming that a parsley-derived polypeptide is expressed in E. coli in an embodiment of the present invention.
  • FIG. 2 is a diagram showing the results of confirming expression of a parsley-derived polypeptide in animal cells in an embodiment of the present invention with a Western blot (A) and a confocal microscope (B).
  • FIG. 3 shows that in an embodiment of the present invention, expression of a parsley-derived polypeptide is increased by LPS (A), and production of nitric oxide is increased (B), and the expression of TLR4 gene by a parsley-derived peptide is It is a diagram as a result of confirming that the gene expression is increased (C) and that the gene expression is suppressed by the TLR4 inhibitor (TAK-242) (D).
  • FIG. 4 shows the TLR4 signaling mechanism by the parsley-derived polypeptide through changes in the expression of the COX2 (A), IL-6 (B) and TNF ⁇ (C) genes, and the increase in the expression of the TLR4 gene is a TLR4 inhibitor (TAK-242). ) Or MAPK inhibitor (PD98059).
  • TLR4 inhibitor TLR4 inhibitor
  • CD80 A
  • MIP2 B
  • C Akt protein
  • IFN- ⁇ (A) in the CD4 + T cells by a parsley-derived polypeptide IFN- ⁇ (B), Mx1 (C), OAS (D), TLR2 (E) and This is a graph of the results of confirming the change in the expression of the TLR4(F) gene.
  • the present invention provides a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2.
  • the polypeptide may be derived from buttercup.
  • the polypeptide is a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2, and may have a size of about 16 kDa.
  • the polypeptide may include variants or fragments of amino acids having different sequences by deletion, insertion, substitution, or a combination of amino acid residues within a range that does not affect the function of the protein. Amino acid exchanges in proteins or peptides that do not totally alter the activity of the molecule are known in the art. In some cases, the polypeptide may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, and the like.
  • polypeptide according to the present invention may also include a polypeptide having 80%, 85%, 90%, 95%, or 98% or more homology with the amino acid sequence described in SEQ ID NO: 2.
  • the present invention may include all polynucleotides encoding the polypeptide.
  • the polynucleotide may be a variant having a different sequence by deletion, insertion, substitution, or a combination of one or more bases within a range encoding a protein having an activity equivalent to that of the polypeptide of the present invention.
  • the nucleotide may be a polynucleotide consisting of the nucleotide sequence described in SEQ ID NO: 1, and all polynucleotides having at least 80%, 85%, 90%, 95%, or 98% homology with the polynucleotide are also included. can do.
  • the present invention provides an expression vector comprising a polynucleotide encoding the polypeptide and a host cell transfected with the expression vector.
  • the polynucleotide may have the characteristics as described above.
  • the polynucleotide may include any polynucleotide encoding the polypeptide of the present invention, and specifically may be a polynucleotide composed of the nucleotide sequence shown in SEQ ID NO: 1.
  • the expression vector refers to a DNA fragment or nucleic acid molecule that is transferred into a cell, and the expression vector refers to a DNA that can be replicated and that a protein can be independently reproduced in a host cell.
  • the recombinant vector into which the polynucleotide encoding the polypeptide of the present invention is inserted is a vector capable of expressing a protein of interest or RNA of interest in a specific host cell, and may contain essential regulatory elements operably linked to express the gene insert. have.
  • the expression vector may include an expression control sequence such as a promoter, an operator, an initiation codon, a stop codon, a polyadenylation signal and an enhancer, and a signal sequence or leader for membrane targeting or secretion Sequence.
  • the expression vector may include a selection marker for selecting a host cell containing the vector, and in the case of a replicable expression vector, the origin of replication may also be included.
  • the expression vector according to the present invention can be prepared in various ways according to the purpose.
  • the expression vector may be a plasmid vector, a cosmid vector, a fosmid vector, a bacteriophage vector, or a viral vector.
  • the host cell may be a host cell genetically modified by the expression vector of the present invention.
  • the polynucleotide or expression vector may exist in the individual as an independent molecule outside the genome within the host cell, or may be stably inserted into the genome of the host cell.
  • the host cell may be a prokaryotic cell or a eukaryotic cell, and may be appropriately selected and used by a person skilled in the art as needed.
  • the eukaryotic cells may be animal cells, for example, the animal cells are immortal hybridoma cells (immortal hybridoma cells), NS/O myeloma cells, 293 cells, Chinese hamster ovary cells (CHO cells), HeLa cells, CapT cells derived from human amniotic fluid, COS cells, and the like may be included.
  • the method of introducing the expression vector of the present invention into a host cell may include all methods known in the art, and specifically, may include a CaCl 2 method, an electroporation method, a microinjection method, and the like.
  • the present invention provides an immunomodulatory agent comprising a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 or a polynucleotide encoding the polypeptide as an active ingredient.
  • the polypeptide or polynucleotide included as an active ingredient in the immunomodulatory agent according to the present invention may have the above-described characteristics.
  • the immunomodulatory agent according to the present invention may contain 10 to 95% by weight of a polypeptide composed of the amino acid sequence described in SEQ ID NO: 2 or a polynucleotide encoding the polypeptide, which is an active ingredient with respect to the total weight of the composition.
  • the immunomodulatory agent of the present invention may further include one or more active ingredients exhibiting the same or similar functions in addition to the active ingredients.
  • the immunomodulatory agent of the present invention may include carriers, diluents, excipients or mixtures thereof commonly used in biological preparations.
  • Any pharmaceutically acceptable carrier may be used as long as it is suitable for delivering the composition in vivo.
  • the carrier is Merck Index, 13th ed., Merck & Co. Inc., saline, sterile water, Ringer's solution, dextrose solution, maltodextrin solution, glycerol, ethanol, or a mixture thereof.
  • conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed.
  • diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants may be added.
  • the immunomodulatory agent of the present invention may be formulated as an oral or parenteral formulation.
  • Oral formulations may include solid formulations and liquid formulations.
  • the solid preparation may be a tablet, a pill, a powder, a granule, a capsule or a troche, and the solid preparation may be prepared by adding at least one excipient to the composition.
  • the excipient may be starch, calcium carbonate, sucrose, lactose, gelatin, or a mixture thereof.
  • the solid preparation may contain a lubricant, and examples thereof include magnesium stearate and talc.
  • the liquid formulation may be a suspension, an inner solution, an emulsion or a syrup. At this time, the liquid formulation may contain excipients such as wetting agents, sweetening agents, fragrances, and preservatives.
  • the parenteral preparation may include injections, suppositories, powders for respiratory inhalation, aerosols for sprays, powders and creams.
  • the injection may include a sterilized aqueous solution, a non-aqueous solvent, a suspension solvent, an emulsion, and the like.
  • the non-aqueous solvent or suspension solvent vegetable oils such as propylene glycol, polyethylene glycol, and olive oil, or injectable esters such as ethyl oleate may be used.
  • the present invention provides an immunomodulatory method comprising administering to an individual a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 or a polynucleotide encoding the polypeptide.
  • polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 used in the immunomodulatory method according to the present invention or a polynucleotide encoding the polypeptide may have the above-described characteristics.
  • the polynucleotide may include any polynucleotide encoding the polypeptide of the present invention, and specifically may be a polynucleotide composed of the nucleotide sequence shown in SEQ ID NO: 1.
  • the individual may be a mammal, and specifically, may be a human.
  • the administration may be oral or parenteral according to the desired method.
  • Parenteral administration may include intraperitoneal, rectal, subcutaneous, intravenous, intramuscular or intrathoracic injection.
  • the administration may be administered in a pharmaceutically effective amount. This may vary depending on the type of disease, the severity, the activity of the drug, the patient's sensitivity to the drug, the administration time, the administration route, the treatment period, and the drugs used at the same time. However, for a desirable effect, the amount of the active ingredient contained in the pharmaceutical composition according to the present invention may be 0.0001 to 1,000 mg/kg, specifically 0.001 to 500 mg/kg.
  • the administration may be once or several times a day.
  • administration may be performed alone or in combination with other therapeutic agents.
  • administration may be sequential or simultaneous.
  • the present invention provides a use of a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 or a polynucleotide encoding the polypeptide for use in the manufacture of a medicament for immunomodulation.
  • polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 or the polynucleotide encoding the polypeptide used in the manufacture of a drug for immunomodulation according to the present invention may have the characteristics as described above.
  • the polynucleotide may include any polynucleotide encoding the polypeptide of the present invention, and specifically may be a polynucleotide composed of the nucleotide sequence shown in SEQ ID NO: 1.
  • the present invention provides an antiviral agent comprising a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 or a polynucleotide encoding the polypeptide as an active ingredient.
  • the polypeptide or polynucleotide included as an active ingredient in the antiviral agent according to the present invention may have the above-described characteristics.
  • the antiviral agent may have the same characteristics as the immunomodulatory agent as described above.
  • the antiviral agent may exhibit antiviral activity by promoting an innate gland immune response that responds most quickly to viral infection. Therefore, the antiviral agent of the present invention can be used to treat all viruses that infect the human body and cause disease in the art.
  • the virus may include all of a single-stranded DNA virus, a double-stranded DNA virus, a single-stranded RNA virus, or a double-stranded RNA virus.
  • the virus is EBV (Epstein-Barr virus), HAV (hepatitis A virus), HBV (hepatitis B virus), HCV (hepatitis C virus), HDV (hepatitis D virus), HEV (hepatitis E virus), lamentation Viruses (Hantaan virus), CMV (cytomegalovirus), HIV (human immunodeficiency virus), flu virus (influenza virus), HPV (human papilloma virus), poliovirus (poliovirus), ebola virus (ebola virus), rotavirus (rotavirus) , Dengue virus, West Nile virus, yellow fever virus, adenovirus, Japanese encephalitis virus, BK virus, smallpox virus smallpox virus), Zika virus, severe febrile thrombocytopenia syndrome virus (SFTS virus), avian influenza virus, retrovirus (retrovirus), or HSV (herpes simplex virus).
  • EBV Epstein-Barr virus
  • HAV hepatitis A
  • the present invention provides a method for preventing, improving, or treating a viral infection comprising administering to an individual a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 or a polynucleotide encoding the polypeptide.
  • the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 or the polynucleotide encoding the polypeptide used in the method for preventing, ameliorating or treating a viral infection according to the present invention may have the characteristics as described above.
  • the polynucleotide may include any polynucleotide encoding the polypeptide of the present invention, and specifically may be a polynucleotide composed of the nucleotide sequence shown in SEQ ID NO: 1.
  • the virus may have the characteristics as described above.
  • the individual may be a mammal, and specifically, may be a human.
  • the administration may have the characteristics as described above.
  • the present invention provides a use of a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 or a polynucleotide encoding the polypeptide for use in the manufacture of a medicament for the prevention, amelioration or treatment of a viral infection.
  • the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 or the polynucleotide encoding the polypeptide used in the manufacture of a medicament for the prevention, amelioration or treatment of a viral infection according to the present invention may have the characteristics as described above.
  • the polynucleotide may include any polynucleotide encoding the polypeptide of the present invention, and specifically may be a polynucleotide composed of the nucleotide sequence shown in SEQ ID NO: 1.
  • the virus may have the characteristics as described above.
  • a base sequence encoding a parsley-derived polypeptide was identified, and an expression vector expressing the identified base sequence was constructed as follows.
  • RNA oligonucleotide linker SEQ ID NO: 3: 5'-AGC AUC GAG UCG GCC UUG UUG GCC UAC UGG-3'
  • T4 ligase T4 ligase
  • the oligo-capped mRNA was isolated using an Oligotex mini kit (Qiagen, USA), and from the isolated mRNA, an Improm-reverse transcription system (Promega, USA) First-stranded cDNA synthesis was performed with a total volume of 20 ⁇ l. Subsequently, the amplified PCR product was treated with SfiI cleavage enzyme, and cDNA having a length of 1.3 kb or more was ligated with the pCNS-D2 vector treated with DraIII cleavage enzyme. The ligated cDNA was transformed into an E. coli Top 10F' (Invitrogen, USA) strain by a conventional electroporation method, and the prepared cDNA library was standardized by a known method.
  • E. coli Top 10F' Invitrogen, USA
  • Colony PCR colony polymerase chain reaction
  • pGEM ® T vector Promega, USA
  • the sequence of the vector in which the PCR product was cloned was analyzed with an ABI DNA sequencer (Applied Biosystems, USA).
  • Example 1 Whether or not the polypeptide derived from parsley identified in Example 1 was expressed in E. coli was confirmed as follows.
  • the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 was amplified using a forward primer (SEQ ID NO: 4: 5'-CCATGGGTGTTCAGAGCCAT-3') and a reverse primer (SEQ ID NO: 5: 5'-CCTCATTGCCAACTACCTCGAG-3').
  • the amplified PCR product was cloned into a pET32a expression vector using NcoI and XhoI cleavage enzymes, thereby preparing a recombinant pET32a-OJPR plasmid.
  • the prepared pET32a-OJPR plasmid was transformed into BL21 strain by electroporation, and cultured in LB (Luria-Bertani) solid medium containing 100 ⁇ g/ml of ampicillin to select desired colonies.
  • the colonies of the BL21 strain transformed with the pET32a-OJPR plasmid were inoculated into LB liquid medium containing ampicillin, and cultured overnight at 37° C. and 200 rpm.
  • the cultured cells were collected and suspended in an LB liquid medium at an OD 600 of 1.0, and IPTG (isopropyl-fD-thiogalactopyranoside) was added, followed by further incubation for 24 hours.
  • the prepared vector was transformed into an E. coli (DH5 ⁇ ) strain, and the transformed strain was cultured in LB medium containing 100 ⁇ g/ml of ampicillin and selected. The selected colonies were inoculated into LB liquid medium containing ampicillin and cultured overnight under conditions of 37° C. and 200 rpm, and cloning of parsley-derived polypeptide was confirmed through sequencing.
  • RAW264.7 cell line using DMEM Dulbecco's Modified Eagle's Medium
  • FBS fetal bovine serum
  • FBS fetal bovine serum
  • 100 U / ml penicillin 100 U / ml penicillin
  • 100 ⁇ g / ml streptomycin 100 ⁇ g / ml streptomycin at 37 °C and 5% CO 2 conditions
  • the prepared RAW264.7 cell line was dispensed into a 6-well plate at about 4 ⁇ 10 5 cells/ml, and further cultured overnight.
  • the confluent of the cultured cells was about 80%, the culture medium was replaced with a serum-free DMEM medium, and the pcDNA3.1(+).OJPR vector was replaced with a PolyFect® transfection reagent kit (Qiagen, USA).
  • the transfected RAW264.7 cell line was cultured in DMEM culture medium containing 500 ⁇ g/ml of G418 (Promega, USA), and cells transfected with pcDNA3.1(+).OJPR vector were selected. The selected cell line was confirmed to be cloned using PCR.
  • a polypeptide derived from parsley was purified by a conventional method.
  • the purified polypeptide was injected into rabbits to prepare a polyclonal antibody against a parsley-derived polypeptide by a conventional method.
  • the Buttercup-derived polypeptide was injected 3 times for 10 weeks or more, and an antiserum specific for the Buttercup-derived polypeptide was obtained in total of 30 ml.
  • the RAW264.7 cell line transfected with pcDNA3.1(+).OJPR vector in Example 3-1 was lysed in 1% RIPC buffer to which a protease and a phosphatase inhibitor were added.
  • the whole cell lysate was electrophoresed on a 10% SDS-PAGE gel, and it was transferred to a PVDF (polyvinylidene difluoride) (BD Bioscience, USA) membrane.
  • TBS-T tris-buffered saline with 0.1% Tween-20
  • the position at which the parsley-derived polypeptide of the present invention is expressed in cells was confirmed with a confocal microscope.
  • the RAW264.7 cell line transfected with pcDNA3.1(+).OJPR vector in Example 3-1 was dispensed into a 4-well chamber slide, and 1 ⁇ g/ml of LPS was present or 5 ⁇ g/ml of purified water parsley derived polypeptide was treated in the absence of it.
  • the cell line was fixed by placing it in 0.1 M phosphate buffer saline (PBS) containing 4% paraformaldehyde for 15 minutes, and then placed in PBS containing 0.25% Triton X-100 (Sigma-Aldrich) and reacted at room temperature for 10 minutes. Made it.
  • PBS phosphate buffer saline
  • Triton X-100 Sigma-Aldrich
  • the cell line after the reaction was pretreated with 0.1% bovine serum albumin (BSA) for 30 minutes at room temperature, and the polypeptide-specific antibody derived from buttercups prepared in Example 2-2 was diluted in PBS buffer containing 1% BSA, and at room temperature. It was reacted for 2 hours.
  • BSA bovine serum albumin
  • the cells were observed with a confocal laser microscope (Olympus FV300, Japan), and at this time, a Largon ion laser with a wavelength of 488 nm and a HeNe laser with a wavelength of 543 nm were used to confirm Alexa Fluor or PI staining, respectively.
  • a confocal laser microscope Olympus FV300, Japan
  • a Largon ion laser with a wavelength of 488 nm and a HeNe laser with a wavelength of 543 nm were used to confirm Alexa Fluor or PI staining, respectively.
  • the results of observation under a microscope are shown in Fig. 2B.
  • the parsley-derived polypeptide was expressed in cells regardless of the presence or absence of LPS.
  • Microarray analysis was performed as follows to see how the parsley-derived polypeptide according to the present invention affects the expression of other genes.
  • biotinylated cRNA was prepared according to the standard Affymetrix protocol. I did. After fragmenting the prepared cRNA, 15 ⁇ g of aRNA was hybridized by reacting with GeneChip Mouse Genome Array (Affymetrix, USA) at 45° C. for 16 hours. Gene chip hybridized with aRNA was washed and stained using Affymetrix Fluidics Station 450. Then, the dyed gene chip was scanned with Affymetrix GeneChip Scanner 3000 7G. The resulting data was analyzed using RMA (Robust Multi-array Analysis).
  • RMA Robot Multi-array Analysis
  • the average of the intensity of each array was arbitrarily set to 100, and the normalized and log-converted intensity values were analyzed using GeneSpring GX12.5 (Agilent Technologies, USA). Changes in gene expression were judged as upregulated genes, which increased by at least 200% compared to the control, and downregulated genes, which decreased by 50% or less compared to the control.
  • Hierarchical clustering data was collected from Genespring GX12.5 that looked similar, and the clustering algorithm was Euclidean distance and average linkage. As a result, the analysis results of the microarray are shown in Table 1 below.
  • HMGB1 high mobility group box 1
  • PKC- ⁇ protein kinase C- ⁇
  • IGFBP4 insulin-like growth factor binding protein 4
  • nucleoporin 85 related to the in vivo immune response was significantly and significantly increased.
  • the protein expressed by the HMGB1 gene is a nuclear protein, which induces an innate immune response through TLR-2 (toll-like receptor-2) and TLR-4 (toll-like receptor-4), and PKC- ⁇ is a TLR It is responsible for intracellular signaling through the -2 mechanism. Accordingly, it was found from the above that the parsley-derived polypeptide of the present invention exhibits an activity to induce an immune response in cells.
  • the RAW264.7 cell line transfected with the parsley-derived polypeptide was dispensed into a 12-well plate at 1.2 ⁇ 10 6 cells per well, and pre-cultured at 37° C. for 3 hours to stably adhere. Thereafter, the cells were washed with PBS, and DMEM medium containing no FBS was added. Here, it was treated with LPS at a concentration of 1, 5 or 10 ⁇ g/ml, cultured, and then RT-PCR was performed using the cultured cells. RT-PCR was performed by a conventional method, and primers described in the nucleotide sequences of SEQ ID NOs: 4 and 5 used in Example 2 were used. The result of confirming the expression level of the obtained PCR product is shown in FIG. 3A. At this time, as a control, a RAW264.7 cell line that was not transfected with anything was used.
  • the expression of the parsley-derived polypeptide was increased depending on the concentration of LPS treated in the cells.
  • a RAW264.7 cell line transfected with a parsley-derived polypeptide of the present invention was treated with LPS at a concentration of 1, 5 or 10 ⁇ g/ml, and nitric oxide production was confirmed using a Grease reagent, and the results are shown in FIG. 3B. Shown in. At this time, as a control, a RAW264.7 cell line that was not transfected with anything was used.
  • the production of nitric oxide in the cell line expressing the parsley-derived polypeptide was increased by about 10 times compared to the control group, which further increased depending on the treatment concentration of LPS.
  • the RAW264.7 cell line which was not transfected with anything, was dispensed into a 12-well plate so as to be 1.2 ⁇ 10 6 per well, and pre-cultured at 37° C. for 3 hours to stably adhere. Thereafter, the cells were washed with PBS, and DMEM medium containing no FBS was added.
  • a parsley-derived polypeptide at a concentration of 1 or 5 ⁇ g/ml was treated, cultured, and then qPCR (quantitative real-time PCR) was performed using the cultured cells.
  • qPCR was performed on a Rotor-gene 6000 instrument (Qiagen, USA) using the SensiMixTM SYBR HiROX kit (Bioline, UK).
  • the reactants for qPCR were prepared including 10 ⁇ l of 2 ⁇ enzyme mastermix, 7 ⁇ l of RNase-free distilled water, 1 ⁇ l of each of the forward and reverse primers, and 1 ⁇ l of the diluted template.
  • the primer for the TLR4 gene was used as a primer consisting of the nucleotide sequence described in SEQ ID NO: 6 (5'-CGCTCTGGCATCATCTTCAT-3') and SEQ ID NO: 7 (5'-TGTTTGCTCAGGATTCGAGG-3').
  • SEQ ID NO: 6 5'-CGCTCTGGCATCATCTTCAT-3'
  • SEQ ID NO: 7 5'-TGTTTGCTCAGGATTCGAGG-3'
  • Pre-degeneration phase 95°C, 15 minutes 1 time Metamorphic stage 95°C, 15 seconds 45 times Annealing step 52°C, 15 seconds Elongation step 72°C, 10 seconds
  • the parsley-derived polypeptide of the present invention significantly increased the expression of the TLR4 gene in cells.
  • the parsley-derived polypeptide was treated with TLR4 inhibitor TAK-242 (0.5, 1, 2 or 5 ⁇ g/ml) in the above process.
  • TAK-242 TAK-242 (0.5, 1, 2 or 5 ⁇ g/ml)
  • QPCR was performed in the same manner as described above.
  • the expression level of the iNOS gene was measured using a primer consisting of the nucleotide sequence described in SEQ ID NO: 8 (5'-TGCCCCTGGAAGTTTCTCTT-3') and SEQ ID NO: 9 (5'-ACTGCCCCAGTTTTTGATCC-3'), and the results are shown in Fig. Shown in 3D.
  • the parsley-derived polypeptide of the present invention increased the expression of the TLR4-dependent genes COX2, IL-6, and TNF ⁇ genes, which were further promoted by LPS treatment.
  • the following experiment was performed to confirm whether the promotion of the TLR4 signaling mechanism by the parsley-derived polypeptide is due to the TLR4/MyD88 signaling cascade. Specifically, the experiment was carried out under the conditions and methods described in Experimental Example 2-3, when a parsley-derived polypeptide was added to the RAW264.7 cell line alone, or when the peptide was treated with TAK-242 or PD98059, a MAP kinase inhibitor. The change in TLR4 gene expression was measured.
  • the RAW264.7 cell line was cultured by treatment with a parsley-derived polypeptide and TAK-242.
  • the cultured cells were fixed by placing them in 0.1 M PBS containing 4% paraformaldehyde for 15 minutes, and washed for 5 minutes using PBS buffer containing 100 mM glycine.
  • the washed cells were placed in PBS containing 0.1% Triton X-100 and reacted at room temperature for 30 minutes.
  • the cell line after the reaction was pretreated with 0.1% bovine serum albumin (BSA) for 30 minutes at room temperature, and the polypeptide-specific antibody derived from buttercups prepared in Example 2-2 was diluted in PBS buffer containing 1% BSA, and at room temperature. It was reacted for 2 hours. After the reaction, the cells were washed 3 times with PBS buffer, and FITC (fluorescein isothiocyanate)-conjugated anti-mouse IgG antibody (Cell Signaling, USA) diluted 1:200 was diluted in 1% BSA and added for 1 hour. It was reacted at room temperature. 5 shows the results of confirming the stained cells using an inverted fluorescent microscope system (Eclipse Ti-S, Japan).
  • BSA bovine serum albumin
  • the RAW264.7 cell line was cultured with a parsley-derived polypeptide and TAK-242, and the cultured cells were used as described in Experimental Example 2-3. And qPCR was performed by the method. At this time, primers for the CD80 and MIP2 genes were used as shown in Table 3 above.
  • the RAW264.7 cell line was dispensed into a 96-well plate at 8 ⁇ 10 4 cells per well and cultured to attach the cells.
  • the cultured cells were co-transfected with 200 ng of pGL4.32 [luc2P/NF- ⁇ B -RE/hygro] plasmid (Promega, USA) and 2 ng of pRL-SV40 plasmid (Promega, USA) by a conventional method. . After 24 hours, the cells were treated with 1 ⁇ g/ml LPS or 5 ⁇ g/ml of parsley-derived polypeptide, followed by further incubation for 6 hours.
  • lucirupase activity was measured using a dual-luciferase reporter assay kit (Bioassay, USA). Specifically, 50 ⁇ l of cell lysate was placed in an opaque white multi-well plate, 100 ⁇ l of Firefly luciferase was added, and the measurement was performed using a luminometer with a wavelength of 565 nm. Then, 100 ⁇ l of Renilla's reagent was added thereto, and the plate was lightly stirred to mix the solution, followed by measurement using a luminometer having a wavelength of 480 nm.
  • the parsley-derived polypeptide of the present invention exhibits antiviral activity while promoting an immune response.
  • it is involved in regulating the antiviral activity or the immune response of the host, or the antiviral genes IFN- ⁇ , IFN- ⁇ , Mx1 (myxovirus resistance protein 1) and OAS ( 2'5'oligoadenylate synthetase 1) gene expression was confirmed.
  • CD4 + T cells were isolated from the spleen of mice, 1 or 5 ⁇ g/ml of parsley-derived polypeptide was treated as described above, and cultured for 24 hours.
  • cDNA was synthesized from the cultured cells, and qPCR was performed as described in Experimental Example 2-3 as a template to confirm the expression of IFN- ⁇ , IFN- ⁇ , Mx1 and OAS genes.
  • the expression of the TLR2 and TLR4 genes was also confirmed.
  • primers for the TLR4 gene were used as primers composed of nucleotide sequences described in SEQ ID NOs: 6 and 7, and primers for other genes were used as shown in Table 4 below.
  • cells treated with anti-CD2 and anti-CD28 were used as a positive control.
  • the parsley-derived polypeptide of the present invention significantly increased the expression of not only IFN- ⁇ , IFN- ⁇ , Mx1 and OAS genes, but also TLR2 and TLR4 genes.
  • the parsley-derived polypeptide according to the present invention can be usefully used as a composition for regulating human immune activity or as an antiviral composition.

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Abstract

La présente invention concerne un polypeptide contenant une séquence nucléotidique spécifique dérivée de l'Oenanthe javanica, et une composition pharmaceutique le comprenant. En particulier, le polypeptide, selon la présente invention, augmente l'expression de divers gènes impliqués dans des réponses immunitaires in vivo, au moyen d'un mécanisme de signalisation TLR, et peut ainsi être utilisé utilement en tant qu'immunomodulateur et, en particulier, peut également être utilisé utilement en tant qu'agent antiviral en favorisant l'expression d'un gène présentant une activité antivirale, parmi les réponses immunitaires.
PCT/KR2020/095011 2019-10-22 2020-03-05 Polypeptide dérivé de l'oenanthe javanica et composition pharmaceutique le comprenant WO2021080400A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7265208B2 (en) * 2001-05-01 2007-09-04 The Regents Of The University Of California Fusion molecules and treatment of IgE-mediated allergic diseases
US7563866B2 (en) * 2002-10-11 2009-07-21 Imvision Gmbh Modular antigen transporter molecules (MAT molecules) for modulating immune reactions, associated constructs, methods and uses
KR20100038968A (ko) * 2008-10-07 2010-04-15 숙명여자대학교산학협력단 실크 펩타이드 및 사이토카인을 유효성분으로 함유하는 것을 특징으로 하는 면역 증강 또는 항암용 조성물
KR20150117741A (ko) * 2014-04-10 2015-10-21 강릉원주대학교산학협력단 청각 유래 면역 증진용 약학적 조성물 및 이의 제조 방법
WO2016161372A1 (fr) * 2015-04-01 2016-10-06 President And Fellows Of Harvard College Immunoconjugués pour la programmation ou reprogrammation de cellules

Family Cites Families (1)

* Cited by examiner, † Cited by third party
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KR100767701B1 (ko) 2004-04-24 2007-10-18 김상희 모노아세틸디아실글리세롤류 화합물을 유효성분으로 함유하는 면역조절제 및 항암제

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7265208B2 (en) * 2001-05-01 2007-09-04 The Regents Of The University Of California Fusion molecules and treatment of IgE-mediated allergic diseases
US7563866B2 (en) * 2002-10-11 2009-07-21 Imvision Gmbh Modular antigen transporter molecules (MAT molecules) for modulating immune reactions, associated constructs, methods and uses
KR20100038968A (ko) * 2008-10-07 2010-04-15 숙명여자대학교산학협력단 실크 펩타이드 및 사이토카인을 유효성분으로 함유하는 것을 특징으로 하는 면역 증강 또는 항암용 조성물
KR20150117741A (ko) * 2014-04-10 2015-10-21 강릉원주대학교산학협력단 청각 유래 면역 증진용 약학적 조성물 및 이의 제조 방법
WO2016161372A1 (fr) * 2015-04-01 2016-10-06 President And Fellows Of Harvard College Immunoconjugués pour la programmation ou reprogrammation de cellules

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